DNA copy number changes in high-grade malignant peripheral nerve sheath tumors by array CGH

<p>Abstract</p> <p>Background</p> <p>Malignant peripheral nerve sheath tumors (MPNSTs) are rare and highly aggressive soft tissue tumors showing complex chromosomal aberrations. In order to identify recurrent chromosomal regions of gain and loss, and thereby novel gene targets of potential importance for MPNST development and/or progression, we have analyzed DNA copy number changes in seven high-grade MPNSTs using microarray-based comparative genomic hybridization (array CGH).</p> <p>Results</p> <p>Considerable more gains than losses were observed, and the most frequent minimal recurrent regions of gain included 1q24.1-q24.2, 1q24.3-q25.1, 8p23.1-p12, 9q34.11-q34.13 and 17q23.2-q25.3, all gained in five of seven samples. The 17q23.2-q25.3 region was gained in all five patients with poor outcome and not in the two patients with disease-free survival. cDNA microarray analysis and quantitative real-time reverse transcription PCR were used to investigate expression of genes located within these regions. The gene lysyl oxidase-like 2 (<it>LOXL2</it>) was identified as a candidate target for the 8p23.1-p12 gain. Within 17q, the genes topoisomerase II-α (<it>TOP2A</it>), ets variant gene 4 (E1A enhancer binding protein, <it>E1AF</it>) (<it>ETV4</it>) and baculoviral IAP repeat-containing 5 (survivin) (<it>BIRC5</it>) showed increased expression in all samples compared to two benign tumors. Increased expression of these genes has previously been associated with poor survival in other malignancies, and for <it>TOP2A</it>, in MPNSTs as well. In addition, we have analyzed the expression of five micro RNAs located within the 17q23.2-q25.3 region, but none of them showed high expression levels compared to the benign tumors.</p> <p>Conclusion</p> <p>Our study shows the potential of using DNA copy number changes obtained by array CGH to predict the prognosis of MPNST patients. Although no clear correlations between the expression level and patient outcome were observed, the genes <it>TOP2A</it>, <it>ETV4 </it>and <it>BIRC5 </it>are interesting candidate targets for the 17q gain associated with poor survival.</p

Integrative Analysis Reveals Relationships of Genetic and Epigenetic Alterations in Osteosarcoma

<div><h3>Background</h3><p>Osteosarcomas are the most common non-haematological primary malignant tumours of bone, and all conventional osteosarcomas are high-grade tumours showing complex genomic aberrations. We have integrated genome-wide genetic and epigenetic profiles from the EuroBoNeT panel of 19 human osteosarcoma cell lines based on microarray technologies.</p> <h3>Principal Findings</h3><p>The cell lines showed complex patterns of DNA copy number changes, where genomic copy number gains were significantly associated with gene-rich regions and losses with gene-poor regions. By integrating the datasets, 350 genes were identified as having two types of aberrations (gain/over-expression, hypo-methylation/over-expression, loss/under-expression or hyper-methylation/under-expression) using a recurrence threshold of 6/19 (>30%) cell lines. The genes showed in general alterations in either DNA copy number or DNA methylation, both within individual samples and across the sample panel. These 350 genes are involved in embryonic skeletal system development and morphogenesis, as well as remodelling of extracellular matrix. The aberrations of three selected genes, <em>CXCL5</em>, <em>DLX5</em> and <em>RUNX2</em>, were validated in five cell lines and five tumour samples using PCR techniques. Several genes were hyper-methylated and under-expressed compared to normal osteoblasts, and expression could be reactivated by demethylation using 5-Aza-2′-deoxycytidine treatment for four genes tested; <em>AKAP12, CXCL5</em>, <em>EFEMP1</em> and <em>IL11RA</em>. Globally, there was as expected a significant positive association between gain and over-expression, loss and under-expression as well as hyper-methylation and under-expression, but gain was also associated with hyper-methylation and under-expression, suggesting that hyper-methylation may oppose the effects of increased copy number for detrimental genes.</p> <h3>Conclusions</h3><p>Integrative analysis of genome-wide genetic and epigenetic alterations identified dependencies and relationships between DNA copy number, DNA methylation and mRNA expression in osteosarcomas, contributing to better understanding of osteosarcoma biology.</p> </div

Data dependencies for two-way combinations.

<p>Heat map plots visualising the odds ratio and significance of data dependencies, with unsupervised hierarchical clustering of the cell lines, for two-way combinations of (A) DNA copy number and gene expression, (B) DNA methylation and mRNA expression and (C) DNA copy number and DNA methylation. The colours of the heat map plot represent the odds ratio for a gene of having one type of aberration given that it has another type of aberration. Green, positive association (odds ratio >1); black, no association (odds ratio = 1) and red, negative association (odds ratio <1). A white circle indicates significance (Benjamini & Hochberg-corrected chi-square p-value <0.05).</p

Hierarchical clustering based on 350 genes that recurrently showed two types of aberrations.

<p>Hierarchical clustering of the osteosarcoma cell lines based on the expression level of the 350 genes that recurrently showed two types of aberrations. The main terms from functional enrichment analysis using DAVID is indicated for each main subcluster of genes. The cell lines are colour-coded in gray and black according to the separation into two main subclusters from the unsupervised hierarchical clustering based on the global mRNA expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048262#pone-0048262-g001" target="_blank">Figure 1A</a>). The cluster was made using Euclidian as distance measure and complete linkage. Green, increased gene expression; red, decreased gene expression.</p

Number of genes with alterations for two-way combinations.

<p>Plot of the number of genes with alterations for all 12 two-way combinations at different sample recurrence thresholds. The recurrence threshold of 6/19 cell lines (>30%) is indicated with a black line.</p

Enrichment analysis of differentially methylated genes using DAVID.

<p>The first five terms in the first three clusters are shown, with enrichment score. The counts and population hits are the number of genes in the gene list and background gene list, respectively, mapping to a specific term. FDR, false discovery rate.</p>*<p>This cluster contained only three terms.</p

Data dependencies for three-way combinations.

<p>Heat map plots visualising the odds ratio and significance of data dependencies, with unsupervised hierarchical clustering of the cell lines, for combinations of hyper-methylation and mRNA expression, conditioning on the DNA copy number status. The colours of the heat map plot represent the odds ratio for a gene of having one type of aberration given that it has another type of aberration. Green, positive association (odds ratio >1); black, no association (odds ratio = 1) and red, negative association (odds ratio <1). A white circle indicates significance (Benjamini & Hochberg-corrected chi-square p-value <0.05). Exp, mRNA expression; CN, DNA copy number.</p

Genomic location of 350 genes that recurrently showed two types of aberrations.

<p>Circos plot showing the genomic location of the 350 genes that recurrently showed two types of aberrations. The locations are colour-coded according to the type of aberrations; orange, hypo-methylation/over-expression; blue, hyper-methylation/under-expression; green, gain/over-expression; red, loss/under-expression; gray, (gain or hypo-methylation)/over-expression and (loss or hyper-methylation)/under-expression. The genomic location of the 14 homeobox genes in the list is indicated in the outermost circle.</p

DNA methylation and mRNA expression of <i>CXCL5.</i>

<p>Plot of the DNA methylation status and mRNA expression level of <i>CXCL5</i> based on methylation-specific PCR and quantitative real-time RT-PCR, respectively, in five cell lines and five tumour samples. The mRNA expression levels have been normalised to the expression of the house-keeping gene <i>GAPDH</i> and then to the average expression level of the two normal osteoblast samples. The DNA methylation status is indicated with coloured circles; black, full methylation, grey, partial methylation. The DNA methylation and mRNA expression levels based on the microarray data are indicated for the cell lines; N, normal methylation; H, hyper-methylation; U, under-expression.</p